báo cáo hóa học: " Anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor, in demyelination of twitcher, a genetic demyelination model" docx

We then treated twi/twi with a daily intraperitoneal injection of ibudilast 10 mg/kg, which suppress TNFα production in the brain.. Results: We found that TNFα-immunoreactive microglia/m

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Research

Anti-inflammatory therapy by ibudilast, a phosphodiesterase

inhibitor, in demyelination of twitcher, a genetic demyelination

model

Kuriko Kagitani-Shimono1,2,3, Ikuko Mohri1,2, Yasushi Fujitani2,

Kinuko Suzuki3, Keiichi Ozono1, Yoshihiro Urade*2 and Masako Taniike*1

Address: 1 Department of Developmental Medicine (Pediatrics), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka

565-0871, Japan, 2 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita, Osaka, 565-0874, Japan and

3 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 919 Brinkhous-Bullitt Bldg, CB7525 Chapel Hill, NC, 27599-7525, USA

Email: Kuriko Kagitani-Shimono - kuriko-shimono@sutv.zaq.ne.jp; Ikuko Mohri - IkukoMohri@aol.com;

Yasushi Fujitani - Fujitani_Yasushi@takeda.co.jp; Kinuko Suzuki - kis@med.unc.edu; Keiichi Ozono - keioz@ped.med.osaka-u.ac.jp;

Yoshihiro Urade* - uradey@obi.or.jp; Masako Taniike* - masako@ped.med.osaka-u.ac.jp

* Corresponding authors

Abstract

Background: Twitcher mouse (twi/twi) is an authentic murine model of Krabbe's disease.

Accumulation of psychosine, resulting in apoptosis of oligodendrocytes and subsequent

demyelination, is a cardinal event to the pathogenesis of this disease Moreover, recruitment of

inflammatory cells plays a significant role in the pathological process in the twi/twi central and

peripheral nervous systems In this study, we investigated the 1) the relationship between tumor

necrosis factor-α (TNFα), pro-inflammatory cytokine, and the progression of this disease and 2)

effect of the anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor

Methods: We quantified the expression level of TNFα and TNF-receptor mRNA in twi/twi using

semi-quantitative RT-PCR The relationship between TNFα expression, apoptosis of

oligodendrocytes and demyelination was studied with immunohistochemistry and TUNEL method

We then treated twi/twi with a daily intraperitoneal injection of ibudilast (10 mg/kg), which suppress

TNFα production in the brain

Results: We found that TNFα-immunoreactive microglia/macrophages appeared in the twi/twi

brain and that the mRNA levels of TNFα and TNF-receptor 1 was increased with the progression

of demyelination The distribution profile of TNFα-immunoreactive microglia/macrophages

overlapped that of TUNEL-positive oligodendrocytes in the twi/twi brain When twi/twi was treated

with ibudilast from PND30, the number of oligodendrocytes undergoing apoptosis was markedly

reduced and demyelination was milder Obvious improvement of clinical symptom was noted in

two of five The failure of constant clinical improvement by ibudilast may result from hepatotoxicity

and/or the inhibition of proliferation of NG2-positive oligodendrocyte precursors

Conclusion: We conclude that anti-inflammatory therapy by a phosphodiesterase inhibitor can be

considered as a novel alternative therapy for Krabbe's disease

Published: 06 April 2005

Journal of Neuroinflammation 2005, 2:10 doi:10.1186/1742-2094-2-10

Received: 04 December 2004 Accepted: 06 April 2005 This article is available from: http://www.jneuroinflammation.com/content/2/1/10

© 2005 Kagitani-Shimono et al; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The twitcher mouse (C57BL/6J-GALC twi ; twi/twi) is a

model of human globoid cell leukodystrophy (Krabbe's

disease), a disorder caused by an inherited deficiency of

the lysosomal enzyme galactosylceramidase [1-3] Twi/twi

shows the symptoms of cerebellar dysfunction such as

action tremor and ataxia around postnatal day (PND) 25,

progressive weight loss after PND 35, and cranial and

peripheral nerve palsy, eventually leading to death around

PND 45 [4,5] Obvious demyelination is recognized after

PND 30 in the central nervous system (CNS)

Cliniconeu-ropathological similarities of this model and the human

disease make this murine model useful for investigations

of pathogenesis as well as for therapeutic approaches [6]

The pathological physiology of twi/twi shares many

com-mon features with that of multiple sclerosis (MS), an

autoimmune demyelinating disease, including the

expres-sion of major histocompatibility complex (MHC)

mole-cules in the CNS [7-9], activation of resident microglia,

recruitment of blood-borne macrophages [10], and the

strong expression of pro-inflammatory cytokines such as

TNFα and interleukin (IL)-6 in the demyelinating focus

[10,11] Therefore, this murine model is useful for

inves-tigating the pathomechanism of demyelination and

devis-ing therapeutic approaches to the neuroinflammation in

general

We previously showed that demyelination of twi/twi was

strongly associated with apoptosis of oligodendrocytes

(OLs) [12] TNFα is the most potent inducer of apoptosis

of OLs among many cytokines in vitro [13] Additionally,

in twi/twi brains, TNFα was reported to be increased in

demyelinating regions [11] and expression of TNFα and

other immune-related molecules were down-regulated in

the pathologically improved regions [10]

Phosphodiesterase inhibitors increase the intracellular

cAMP levels and reduce the inflammatory cytokines such

as TNFα in vitro [14] Ibudilast, a non-selective

phos-phodiesterase inhibitor, was reported to reduce

demyeli-nation in experimental allergic encephalomyelitis (EAE)

and to suppress TNFα production by microglia in vitro

[15,16]

In this study we found that 1) the expression of TNFα and

its receptor TNF-R1 was associated with demyelination

and that 2) ibudilast could reduce demyelination and

alleviate the progression of disease and suppress TNFα

production in twitcher brain These results were

consist-ent with the hypothesis that TNFα signaling enhances

apoptosis of OLs and demyelination in twi/twi, and

sug-gested that suppression of inflammation may provide

new therapeutic approaches to demyelinating diseases

Methods

Animals

All animal experiments were performed according to the Guidelines for the Protection of Experimental Animals issued by the Japanese Government, the US National Institutes of Health, and the Society for Neuroscience

Heterozygous breeder pairs of twitcher (twi/+) were

origi-nally purchased from Jackson Laboratory (Bar Harbor,

ME) Twi/twi and normal age-matched siblings (+/+) were

identified by genotyping with genomic DNA extracted from the clipped tails by use of a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) Genotyping was performed as previously reported [17]

Materials

The following primary antibodies were used: phycoeryth-rin (PE)-conjugated anti-TNFα (1:50; PharMingen, San Diego, CA), mouse monoclonal anti-myelin basic protein (MBP) antibody (1:200; Sternberger Monoclonals Incor-porated, Lutherville, MA), rabbit polyclonal anti-rat-pi-form of glutathione-S-transferase (pi-GST) antibody (1:1000; MBL, Nagoya, Japan), rabbit polyclonal anti-cow glial fibrillary acidic protein (GFAP) antibody (prediluted;

DAKO, Glostrup, Denmark), biotinylated Ricinus commu-nis-agglutinin-1 (RCA-1) (50 µg/ml; Vector Laboratories,

Burlingame, CA), and rabbit polyclonal NG2 chondroitin sulfate proteoglycan (NG2) antibody (1:200; Chemicon

International Inc., Temecula, CA) Biotinylated Ricinus communis-agglutinin-1 (RCA-1) (50 µg/ml) was pur-chased from Vector Laboratories (Burlingane, CA)

Tissue preparation

Brains from twi/twi and +/+ mice killed at PND 20, 30, and

40 (n = 3 for each timing period) were immunostained for TNFα The mice were perfused with cold physiological saline under deep inhalation anesthesia with sevoflurane, and the isolated brains were quickly frozen in liquid nitro-gen For routine histochemical staining, mice (n = 3 for each groups) were perfused with physiological saline, fol-lowed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) The brain was removed, postfixed and embedded in paraffin blocks Luxol fast blue (LFB)-peri-odic acid Schiff (PAS) staining was performed on the

par-affin sections of twi/twi and +/+ at PND 40 for evaluation

of neuropathology

For the determination of mRNA levels, groups of twi/twi and +/+ (n = 3 each timing period) were killed at PND 20,

30, and 40 under appropriate anesthesia The brains were then removed, divided into the cerebrum and cerebellum/ brain stem, and quickly frozen in liquid nitrogen

Immunocytochemistry

Frozen sections were fixed at 4°C in acetone and incu-bated with PE-conjugated rat anti-mouse TNFα antibody

for 48 h For double labeling with RCA-1 and anti-TNFα,

TNFα-stained sections were reacted with biotinylated

RCA-1 for 30 min at room temperature, and then with

avi-din-D-fluorescein isothiocyanate isomer (avidin-FITC;

Vector Laboratories), diluted 1:1000 with PBS, for 30 min

For NG2 immunostaining, after blocking with 0.3%

Tri-ton-X100 for 1 h, frozen sections were incubated with

anti-NG2 antibody for 12 h at 4°C, and incubated with

Alexa 488-conjugated anti-rabbit IgG (H+L) (1:400;

Molecular Probes, Inc., Eugene, OR) for 2 h

Paraffin sections were used for immunostaining for MBP

and pi-GST, and terminal deoxynucleotidyltransferase

(TdT)-mediated dUTP nick end labeling (TUNEL) For

immunocytochemistry, sections on glass slides were

incu-bated serially with mouse anti-MBP or rabbit anti-pi-GST

antibody, biotinylated goat anti-mouse or anti-rabbit

immunoglobulins (Vector Laboratories), and

avidin-biotin complex by using an ABC elite kit (ABC; Vector

Laboratories) Immunoreactions were visualized by

immersing the slides in a 0.03% H2O2 solution in 50 mM

Tris-HCl (pH 7.6) containing 0.05% diaminobenzidine

tetrahydrochloride (DAB) and 0.25% nickel ammonium

sulfate Twi/twi and +/+ at PND 40 were subjected to

TUNEL staining Nuclei with DNA fragmentation were

detected by using an in situ apoptosis detection kit (Takara

Biomedicals, Osaka, Japan) Briefly, after pretreatment

with 0.1% trypsin for 15 min at 37°C, sections were

reacted with TdT, dNTPs, and FITC-labeled dUTP for 90

min at 37°C, followed by horseradish peroxidase

(HRP)-conjugated anti-FITC antibody overnight at 4°C The

immunoproduct was visualized with the same protocol

described above

To identify the type of TUNEL-positive cells, we combined

the staining for pi-GST, GFAP and RCA-1 with the TUNEL

procedure After TUNEL staining, sections were incubated

with PBS containing 0.3% TritonX-100 and 10% normal

goat serum for 30 min and then with rabbit anti rat-pi GST

antibody, rabbit anti-cow GFAP antibody or biotinylated

RCA-1 at 4°C overnight The procedures were basically

the same as described above except for the use of

ABC-alkaline phosphatase and naphthol AS-BI phosphate

cou-pled with hexazotized new fuchsin (Merck, Darmstadt,

Germany) as a chromogen

Quantification of the level of TNFα-mRNA

Total RNA was isolated from the quick-frozen brains with

Isogen (Nippon gene, Toyama, Japan) The random

9-mers-primed cDNA was prepared with an RNA-LA-PCR

Kit (Takara Shuzo, Kyoto, Japan) and 2 µg of total RNA

A LightCycler PCR and detection system (Roche

Diagno-sis, Mannheim, Germany) was used for the amplification

and quantification of mRNA for TNFα, TNFR1, TNFR2

and glycerol aldehyde-3-phosphate dehydrogenase (G3PDH) as previously described [18] G3PDH served as

an internal control The sequence-specific primers used were as follow: TNFα forward primer: 5'-AGT-GACAAGCCTGTAGCCCACG-3', TNFα reverse primer: 5'-TTTCTCCTGGTATGAGATAGC-3', TNFR1 forward primer: 5'-CTAAACAGCAGAACCGAGTGT-3', TNFR1 reverse primer: 5'-AGATACGTAGAGTGTCCTTGG-3', TNFR2 forward primer: 5'-ATAAAGCCACAC-CCACAACCT-3', TNFR2 reverse primer: CATCTCCCT-GCCACTCACAA-3', G3PDH forward primer: 5'-TGAACGGGAAGCTCACTGG-3', and G3PDH reverse primer: 5'-TCCACCACCCTGTTGCTGTA-3' The con-structs, used to create a standard curve, were made by cloning each amplified fragment into the Hind III site of

a pGEM vector (Promega, Madison, WI) The number of copies was calculated by plotting a dilution series on this standard curve in each PCR experiment For amplification detection, the LightCycler DNA Master Hybridization Probes Kit was used Quantification of TNFα mRNA was performed by conducting 50 cycles of repeated denatura-tion (1 s at 89°C), annealing (5 s at 58°C), and enzymatic chain extension (10 s at 72°C) The PCR amplification conditions for G3PDH were 40 cycles of repeated denatur-ation (1 s at 87°C), annealing (5 s at 57°C), and enzy-matic chain extension (10 s at 72°C) Quantification of TNFR1 and TNFR2 mRNAs was made by using 50 cycles

of repeated denaturation (1 s at 89°C), annealing (5 s at 58°C), and enzymatic chain extension (10 s at 72°C) Duplicated PCR products were evaluated by melting curve analysis

Administration of Ibudilast

Ibudilast was a generous gift from Kyorin Pharmaceutical

Co Ltd (Tokyo, Japan) After dissolved to a concentration

of 1 mg/ml in physiological saline containing 10% v/v of polyoxyethylene hydrogenated castor oil 60 (HCO60), ibudilast (10 mg/kg) was injected intraperitoneally daily

into three twi/twi from PND 15 to PND 40, and five twi/ twi from PND 30 to PND 45 For controls, the same vol-ume of HCO 60 was injected into two twi/twi from PND

15 to PND 40 and four twi/twi from PND 30 to PND 45.

The density of TUNEL-positive cells in the demyelinating

lesion in twi/twi, treated from PND 30 to PND 45 was

cal-culated by using MacSCOPE software (Mitani Co, Fukui, Japan) Two independent neuropathologists examined the LFB-PAS-stained coronal sections (four sections per mouse) at the level of the optic chiasm and at the cerebel-lopontine angles containing the paraflocculus in a dou-ble-blind manner and scored the severity of demyelination from 0 to 5 0: no demyelination, 1: slight demyelination, 2: less than 25% of the areas are occupied

by a demyelination focus, 3: 25% ~ 50% of the areas occu-pied, 4: 50 ~ 75% of the areas occuoccu-pied, 5: more than 75%

of the areas occupied The scores were average of two

examiners' evaluations

In situ hybridization for TNFα

The cDNA probe for TNFα comprised a 268-bp PCR

frag-ment (forward primer;

GATGGGTTGTACCTTGTC-TACTCC-3' and reverse primer;

5'-CTAAGTACTTGGGCAGATTGACCT-3') from the mouse

TNFα, and was subcloned into a pGEM-T Easy vector

(Promega, Madison, Wisconsin) In situ hybridization was

carried out by using manual capillary action technology

with a Microprobe staining system (Fisher Scientific

Inter-national, Hampton, NH) as previously described [19,20]

First, brain sections (10 µm) were deparaffinized with

Auto Dewaxer (Research Genetics, Huntsville, AL) The

sections were rinsed in Auto Alcohol, Universal Buffer,

and Immuno/DNA buffer (Research Genetics)

Prediges-tion by proteinase K (15 µg/ml; Sigma-Aldrich, St Louis,

MO) was performed to increase the tissue penetration of

the probe After this digestion, the tissue sections were

treated with Immuno/DNA buffer The DIG-labeled cRNA

probe was diluted to 0.5 µg/ml with Brigati probe diluent

(Research Genetics), 50% deionized formamide, and 50%

dextran sulfate The probe solution was heated at 90°C to

denature the cRNA structures and applied to the slides

The hybridization of tissue and probe was done at 50°C

for three hours After hybridization, the slides were

washed in 2 × SSC containing nonionic detergent The

detection of the DIG-labeled RNA was performed by using

the Genius DNA labeling and detection kit (Roche

Diag-nostics) For counterstaining, neutral red was applied

Statistical analysis

Student's t test was performed by using Stat View software

(SAS Institute, Cary, NC) p< 0.05 was considered as

significant

Results

Levels of TNFα and TNFR1 are increased in the twitcher

cerebellum

The level of TNFα mRNA was the same in both cerebellum

and cerebrum of the +/+ at any age examined In the

cere-brum, the level of TNFα-mRNA in twi/twi was almost the

same as that in +/+ until PND 30, however, it increased to

become approximately 15 times higher at PND 40 than

that of +/+ In the cerebellum, there was no difference in

the TNFα mRNA level between twi/twi and +/+ at PND 20,

however, its level increased significantly in twi/twi after

PND 30, becoming 40 times higher in twi/twi than +/+ at

PND 40 (Fig 1A)

Next, we investigated the levels of TNFR1 and TNFR2 In

the +/+ cerebellum, the level of TNFR1 mRNA was

con-stant throughout all the ages examined, whereas in the

twi/twi cerebellum, it significantly increased with the

pro-gression of demyelination, becoming 50 times higher

than that in +/+ at PND 40 In contrast, mRNA for TNFR2 increased in twi/twi only after PND 40, when compared with that for +/+ (Fig 1B).

Immunocytochemical analysis revealed that TNF α-immu-noreactive cells were not recognized at PND 20 (Fig 1C)

in twi/twi However, many TNFα-immunoreactive cells were found in the cerebral white matter, brain stem and cerebellar white matter (CWM) at PND 30 (Fig 1D) and

40 (Fig 1E) On the other hand, TNFα-immunoreactive

cells were not detected anywhere in the +/+brain even at

PND 40 (Fig 1F) These data were compatible with the data of the quantitative RT-PCR

TNFα expression is increased in microglia/macrophages within demyelinating lesions in twi/twi

The morphological characteristics of TNFα-positive cells were an irregular cellular contour and lack of delicate processes, reminiscent of ameboid microglia/macro-phages Furthermore, TNFα-positive cells were positive for RCA-1, a marker for macrophage (arrows in Fig 2A), but negative for pi-GST, a marker for OLs, or GFAP, a marker for astrocytes (data not shown), confirming those

cells to be microglia/macrophages In the twi/twi brain,

both TNFα-positive cells and TUNEL-positive cells were most abundant in the CWM (Fig 2B, C) and in the spinal trigeminal tract (sp5) in the superior midbrain (Fig 2E, F) The majority of TUNEL-positive cells were also positive for pi-GST (arrowheads in Fig 2C, F, I), identifying them

as OLs (inset in Fig 2C) These lesions of the cerebellum were most severely demyelinated judged by MBP immu-nostaining (Fig 2D, G) In contrast, in the corpus callo-sum, where demyelination was milder than in the cerebellum, only a few TNFα-positive cells were detected (Fig 2H – J)

Administration of phosphodiesterase inhibitor ameliorates demyelination and the clinical symptoms

To investigate whether the inflammatory response in microglia/macrophages contributes to the demyelination

in twi/twi, we administered a phosphodiesterase inhibitor, ibudilast, to twi/twi Two out of five twi/twi treated from

PND 30 revealed strikingly milder clinical symptoms (Fig

3A) Even at PND 45, two of ibudilast-treated twi/twi from

PND 30 could move smoothly despite mild hindlimb paralysis, and showed less severe tremor and ataxia than

vehicle-treated twi/twi These mice were bigger than vehi-cle-treated twi/twi, as they had less weight loss (Fig 3B) In contrast, ibudilast-treated twi/twi from PND 15 showed

neither apparent clinical improvement nor elongation of lifespan, however, their body weights were heavier than

those of vehicle-treated twi/twi.

TNFα and its receptors increased as demyelination proceeded

Figure 1

TNFα and its receptors increased as demyelination proceeded A-B: Quantification of mRNA for TNFα (A) and its receptors (B) The copies of mRNA for TNFα have increased in twi/twi (■) after PND 30, especially in the cerebellum, when compared with those in +/+ (▲) Those for TNFR1 in the cerebellum have increased in twi/twi after PND 30 The copies of mRNA for TNFR2 have increased in twi/twi only after PND 40, when compared with those for +/+, but the difference was not significant

(B) Bar represents mean ± SE * p < 0.01 C-F: TNFα immunostaining in the cerebellum There are no TNFα-positive cells in

the cerebellum of twi/twi mice at PND 20 (C) Immunoreactive cells for TNF α are progressively increased in number in the twi/ twi cerebellar white matter between PND 30 (D) and PND 40 (E) In contrast, there are no TNFα positive cells in +/+ brains

at any ages examined (F) Tw and W represent twi/twi and wild-type mice, respectively The data represent mean ± SE IG:

internal granular layer, CWM: cerebellar white matter Scale bar = 50 µm

TNFα is expressed in activated microglia/macrophages in the regions where many apoptotic OLs are recognized with severe demyelination

Figure 2

TNFα is expressed in activated microglia/macrophages in the regions where many apoptotic OLs are recognized with severe demyelination A: Double labeling of TNFα and RCA-1 of the twi/twi cerebrum at PND 40 Arrows indicate

microglia/macro-phages, which are double positive for TNFα and RCA-1 B-J : In twi/twi at PND 40, there are many TNFα-positive cells (B, E) as

well as many TUNEL-positive cells (C, F) in the CWM and sp5, where severe demyelination was present as judged from the results of MBP immunostaining (D, G) These apoptotic cells are immunostained with pi-GST, identified to be OLs (inset in C)

In the corpus callosum (cc), there are only a few TNFα-positive cells (H) and TUNEL-positive cells (I), where demyelination was milder than in the cerebellum (J) Asterisks and double asterisks represent the same region in the serial sections Scale bars

= 50 µm (B-J), 10 µm (inset in "C")

The signal for TNFα mRNA obtained by in situ

hybridiza-tion was recognized in the cells with small nuclei in the

CWM and sp5 of vehicle-treated twi/twi (inset in Fig 4A),

corresponding to the presence of TNFα-immunoreactivity

in the microglia This signal was significantly reduced in

the ibudilast-treated twi/twi (Fig 4B, D) The number of

TUNEL-positive cells was decreased in the CWM in

ibudi-last-treated twi/twi (Fig 4F, H) compared with that of the

vehicle-treated mice (Fig 4E, G) TUNEL-positive cells were decreased in other regions such as the 8th nerve (8 n)

and sp5 in ibudilast-treated twi/twi than in vehicle-treated

mice (Fig 5, the upper bar graph)

LFB-PAS staining revealed that the demyelination was remarkably suppressed in the ibudilast-treated mice from PND 30 (Fig 4J, L) compared with the vehicle-treated ones (Fig 4I, K), as shown in the score of demyelination (Fig 5, lower bar graph) From these lines of evidence, we concluded that the demyelination and clinical symptoms were reduced with inhibition of TNFα in twi/twi.

Ibudilast treatment decreased NG2-positive OL progenitors

To evaluate the effect of ibudilast to the OL progenitors, frozen sections were stained with anti-NG2 antibody In

contrast to the vehicle-treated twi/twi, ibudilast-treated twi/twi showed fewer NG2-positive OL progenitors (Fig.

6), suggesting that incomplete clinical improvement may result from the insufficient remyelination in

ibudilast-treated twi/twi.

Discussion

Our results suggested that secondary inflammation via TNFα produced in microglia/macrophages remarkably enhances the apoptosis of OLs and aggravates the

demy-elination due to the metabolic defect in twi/twi These are

consistent with previous reports showing that TNFα

induces apoptosis of OLs in vitro [21,22], and that TNFα

is upregulated in macrophages and globoid cells in twi/twi

[11]

TNFα is a well-established pro-inflammatory mediator of immune process, and is essential to the maintenance of CNS homeostasis However, its overexpression leads to the development of chronic CNS inflammation and degeneration [23] We previously observed emergence of TNFα-expressing cells with progression of demyelination and the number of those cells declined following bone

marrow transplantation with prolonged survival in twi/twi

[10] TNFα was expressed by infiltrating blood mononu-clear cells, and its expression was well correlated with the extent of demyelination in another genetic demyelinating disease, X-linked adrenoleukodystrophy[24], and in the

MS [25] TNFα-transgenic mice showed more severe demyelination and macrophage infiltration in EAE, a mouse model for MS [26] Of two TNFRs, TNFR1 was reported to mediate the pathogenetic effects of TNFα, such as inflammation, cytotoxicity, and apoptosis of OLs

in EAE [13,27-29] Our study showed that TNFR1 was dominant from the early demyelinating stage and that

A: Two twi/twi at PND 44, one ibudilast-treated and other

vehicle-treated from PND 30

Figure 3

A: Two twi/twi at PND 44, one ibudilast-treated and other

vehicle-treated from PND 30 The ibudilast-treated twi/twi is

much bigger and can walk faster and reach the feedbox, in

spite of mild paralysis and spasticity in lower limbs In

con-trast, the vehicle-treated twi/twi can no longer walk nor feed

itself In addition, the ibudilast-treated twi/twi has much

milder tremor than the vehicle-treated twi/twi B: The change

of body weight (g) of ibudilast- and vehicle-treated twi/twi

Both twi/twi treated with ibudilast or vehicle from PND 15

(●: ibudilast-treated twi/twi, ❍: vehicle-treated twi/twi)

showed less weight gain compared with those treated from

PND 30 (■: ibudilast-treated twi/twi, 䊐: vehicle-treated twi/

twi), and no prolongation of the life span However,

ibudilast-treated twi/twi showed less body weight loss than

vehicle-treated twi/twi N = 3 and 2 in ibudilast- and vehicle-vehicle-treated

twi/twi from PND 15 The ibudilast-treated twi/twi from PND

30 were bigger and showed milder clinical detrerioration N

= 5 and 4 in ibudilast- and vehicle-treated twi/twi from PND

30 The data represent mean ± SE

Suppression of TNF mRNA expression is accompanied by inhibition of apoptosis and subsequent milder demyelination in

ibud-ilast-treated twi/twi at PND45

Figure 4

Suppression of TNF mRNA expression is accompanied by inhibition of apoptosis and subsequent milder demyelination in

ibudilast-treated twi/twi at PND45 A, B, E, F, I, J: CWM, C, D, G, H, K, L: sp5 A-D: In situ hybridization of TNFα mRNA in

vehicle-treated twi/twi (A, C) and ibudilast-treated twi/twi (B, D) Whereas vehicle-treated twi/twi show abundant signals in

CWM (A) and sp5 (C), TNFα mRNA signals are remarkably reduced in the ibudilast-treated twi/twi (B, D) Inset in "A" shows

TNF-α mRNA-positive microglia E-H: TUNEL staining of vehicle-treated twi/twi (E, G) and ibudilast-treated twi/twi (F, H) Ibud-ilast-treated twi/twi shows fewer positive cells than are seen in vehicle-treated twi/twi Arrowheads indicate TUNEL-positive cells I-L: LFB-PAS staining of vehicle-treated twi/twi (I, K) and ibudilast-treated twi/twi (J, L) In the ibudilast-treated twi/ twi, CWM and sp5 show much milder demyelination than in vehicle-treated twi/twi Scale bar = 100 µm (I-L), 50 µm (A-H), 10

µm (inset in "A")

α

demyelination and OL apoptosis was alleviated by the

suppression of TNFα in ibudilast-treated twi/twi These

lines of evidence suggested that the stimulation of TNFR1

was associated with apoptosis of OLs and demyelination

in twi/twi Therefore, we believe that

TNFα/TNFR1-medi-ated secondary inflammation is involved in the

progres-sion of pathology in varieties of demyelinating diseases

In this study, we selected ibudilast as an

immunomodula-tory agent which also suppressed the production of other

inflammatory mediators, such as nitric oxide (NO),

IFN-γ, and IL-6, and enhanced the production of the inhibitory

cytokine, IL-10, and neurotrophic factors, including nerve

growth factor (NGF), glia-derived neurotrophic factor

(GDNF) and neurotrophin (NT-4) [30] Since inducible

nitric oxide (iNOS) and IL-6 were strongly upregulated in

twi/twi and Krabbe's disease [10,11,31], the positive effect

of ibudilast may be also associated with suppression of

iNOS and IL-6, and enhancement of inhibitory cytokines

and neurotrophic factors However, taking into account that TNFα is the most potent cytotoxic cytokine, and that signals for TNFα mRNA were remarkably suppressed in

the areas of severe demyelination in ibudilast-treated twi/ twi, the effect of ibudilast may be mediated, at leaset in

part, by the suppression of TNFα expression

Several different types of anti-TNFα therapy have been recently reported For example, TNF-receptor-p55-immu-noglobulin fusion protein was reported to suppress demy-elination in EAE [32,33], whereas it showed no significant efficacy in MS patients [34,35] Infliximab and etanercept, used as anti-TNFα agents for rheumatoid arthritis and Crohn's disease, are rather reported to induce demyelina-tion [36,37] In contrast to the poor outcomes of these direct TNFα suppression, interferon (IFN) β [38,39] and glatiramer acetate (GA) [40,41] have been widely approved as effective immunomodulatory treatments for

MS TNFα production was significantly reduced in mono-cytes from patients treated by GA [42], which acts prima-rily as an antigen for T lymphocytes Furthermore, MS patients who received administration of IFNβ revealed decreased mRNA for TNFα [43] and an increase in serum TNFRs, of which TNFR2 may play a protective role for myelin [44]

The clinical symptoms were improved in only two

ibudi-last-treated twi/twi, whereas the demyelination was milder

in all of the treated twi/twi In the ibudilast-treated twi/twi

without clinical improvement, the number of NG2-immunoreactive OL progenitors was decreased, compared

with that in vehicle-treated twi/twi Lack of TNFα has been reported to result in a significant delay of remyelination in

a cuprizone-induced demyelination model, due to a reduced number of proliferating OL progenitors [45], since the signal transduction of TNFα via p75 TNF recep-tor 2 (TNFR2) is known to induce proliferation of OL pro-genitors [27,28] Therefore, TNFα stimulation may be involved not only in the apoptotic signal pathway medi-ated by TNFR1, but may also play a regenerative role via activation of TNFR2 [46] Earlier treatment with ibudilast from PND 15 showed less apparent clinical effect com-pared with that from PND30, probably due to the follow-ing two reasons: daily intraperitoneal injection itself

could be too invasive for younger twi/twi to gain weight

and/or TNFR2-stimulated proliferation of OLs in this period of active myelination is profoundly inhibited by the reduced TNFα production These lines of evidence suggested that TNFα inhibitor should be used for a lim-ited period of time or in a TNFR1-specific manner The cytotoxicity of ibudilast may be another explanation for the failure of clinical improvement in some cases: when we administered a high dosage (20 mg/kg) of

ibud-ilast to twi/twi, it induced vacuolar degeneration of

Ibudilast-treated twi/twi show pathological improvement

Figure 5

Ibudilast-treated twi/twi show pathological improvement

Population of TUNEL-positive cells and neuropathological

scores of LFB-PAS in ibudilast- (closed-boxed; N = 4) or

vehicle-treated (hatched; N = 3) twi/twi In CWM, 8 n, and

sp5 of the ibudilast-treated twi/twi, the number of

TUNEL-positive cells is decreased to half of those in the

vehicle-treated twi/twi They also recognized significantly milder

demyelination in LFB-PAS stain 8 n: the 8th nerve *p < 0.01,

**p < 0.05 The error bars represented standard deviations

hepatocytes and the mice died of the hepatic failure (data

not shown) When ibudilast was directly administered by

an intraventricular injection to avoid systemic adverse

effect, periventricular tissues were extensively damaged by

this chemical These results indicate that other drugs with

less cytotoxicity are necessary to improve the symptoms of

twi/twi and other demyelination diseases.

From these lines of evidence, we propose that

anti-inflam-matory therapy by a phosphodiesterase inhibitor during

an appropriate period, may be a reliable supportive

treat-ment for Krabbe's disease for which there is no effective

treatment except bone marrow transplantation

[6,23,47-49]

Conclusion

These results suggest that the suppression of

inflamma-tion by a phosphodiesterase inhibitor could be a novel

therapy in genetic demyelination

List of abbreviations

twitcher mouse (twi/twi)

tumor necrosis factor-α (TNFα)

postnatal day (PND)

central nervous system (CNS) multiple sclerosis (MS) major histocompatibility complex (MHC) interleukin (IL)

oligodendrocytes (OLs) experimental allergic encephalomyelitis (EAE) phycoerythrin (PE)

myelin basic protein (MBP) pi-form of glutathione-S-transferase (pi-GST) glial fibrillary acidic protein (GFAP)

Ricinus communis-agglutinin-1 (RCA-1)

phosphate buffer (PB) fluorescein isothiocyanate isomer (FITC)

Ibudilast surpresses proliferation of NG2-positive OL progenitors

Figure 6

Ibudilast surpresses proliferation of NG2-positive OL progenitors A: Vehicle-treated twi/twi shows many NG2-positive OL progenitors B: Ibudilast-treated twi/twi shows decreased number of NG2-positive OL progenitors Allows: NG2-positive OL

progenitors labeled with Alexa 488 Scale bar = 50 µm